Engagement of the B cell antigen receptor (BCR) by polyvalent ligands initiates two simultaneous processes, cell signaling and receptor internalization. Signals transmitted through the BCR activate a myriad of signaling pathways which integrate to control the transcriptional and cell biological machineries that determine cellular responses. Internalization of the receptor, and the antigen bound to it, is required for the effective presentation of MHC class II restricted peptides to T cells. Both receptor signaling and the recruitment of T cell help are required for normal humoral immune responses and both are often aberrant in autoimmunity. As important as these two processes are, the relationships between them are poorly understood. It has been postulated that antigen receptor internalization may limit signaling by targeting activated receptor complexes for degradation. Conversely, it has been suggested that internalization of the BCR is required for the activation of some MAP kinase pathways. Much of the confusion regarding the impact of BCR internalization on signaling is because the mechanisms regulating internalization are not known. We have demonstrated that within Ig?, the non-ITAM tyrosines are the principal determiners of internalization. This observation suggested that, following receptor engagement, phosphorylated receptor complexes were retained on the cell surface while only non-phosphorylated receptors were internalized. Using direct biochemical assays, we demonstrated that this was indeed the case. Based on these studies, in this grant application we propose a model in which receptor endocytosis does not directly regulate signal propagation. Rather, we hypothesize that endocytosis modulates signal amplitude by removing non-phosphorylated receptor complexes from the cell surface. This model will be tested in the following Specific Aims. Aim 1. Derive a quantitative model of receptor signaling and internalization. In Aim 1A, we will examine how BCR internalization modulates the extent of receptor phosphorylation and the activation of downstream signaling pathways. For these experiments, we will use a DT40 cell line in which the endogenous clathrin alleles have been disrupted and replaced with a human clathrin complementary DNA under the control of a tetracycline-regulated promoter. In Aim 1B, we will use the quantitative data obtained from the experiments in 1A to develop a mathematical model of the relationships between receptor signaling and internalization. Aim 2. To identify the linkers which recognize the tyrosine-based internalization motifs in Ig?. We will concentrate our efforts on Ig? Y176 and Y204 because they are important determiners of receptor internalization. These two tyrosines are the same residues that, in combination, recruit BLNK to the receptor complex. Both peptide affinity precipitations and immunoprecipitations from wild-type and mutant receptors will be used to isolate possible adaptor proteins. These will be characterized using HPLC coupled with ion trap mass spectrometry. Putative targets will be validated in co-immunoprecipitation and knockdown experiments. [unreadable] [unreadable]